Drosophila Epsin's role in Notch ligand cells requires three Epsin protein functions: The lipid binding function of the ENTH domain, a single Ubiquitin interaction motif, and a subset of the C-terminal protein binding modules

Section of Molecular Cell and Developmental Biology, Institute for Cell and Molecular Biology, The University of Texas at Austin, Austin, TX 78712, USA.
Developmental Biology (Impact Factor: 3.55). 03/2012; 363(2):399-412. DOI: 10.1016/j.ydbio.2012.01.004
Source: PubMed

ABSTRACT Epsin is an endocytic protein that binds Clathrin, the plasma membrane, Ubiquitin, and also a variety of other endocytic proteins through well-characterized motifs. Although Epsin is a general endocytic factor, genetic analysis in Drosophila and mice revealed that Epsin is essential specifically for internalization of ubiquitinated transmembrane ligands of the Notch receptor, a process required for Notch activation. Epsin's mechanism of function is complex and context-dependent. Consequently, how Epsin promotes ligand endocytosis and thus Notch signaling is unclear, as is why Notch signaling is uniquely dependent on Epsin. Here, by generating Drosophila lines containing transgenes that express a variety of different Epsin deletion and substitution variants, we tested each of the five protein or lipid interaction modules for a role in Notch activation by each of the two ligands, Serrate and Delta. There are five main results of this work that impact present thinking about the role of Epsin in ligand cells. First, we discovered that deletion or mutation of both UIMs destroyed Epsin's function in Notch signaling and had a greater negative impact on Epsin activity than removal of any other module type. Second, only one of Epsin's two UIMs was essential. Third, the lipid-binding function of the ENTH domain was required only for maximal Epsin activity. Fourth, although the C-terminal Epsin modules that interact with Clathrin, the adapter protein complex AP-2, or endocytic accessory proteins were necessary collectively for Epsin activity, their functions were highly redundant; most unexpected was the finding that Epsin's Clathrin binding motifs were dispensable. Finally, we found that signaling from either ligand, Serrate or Delta, required the same Epsin modules. All of these observations are consistent with a model where Epsin's essential function in ligand cells is to link ubiquitinated Notch ligands to Clathrin-coated vesicles through other Clathrin adapter proteins. We propose that Epsin's specificity for Notch signaling simply reflects its unique ability to interact with the plasma membrane, Ubiquitin, and proteins that bind Clathrin.

170 Reads
  • Source
    • "The requirement for the UIM in epsin1-mediated receptor transport is echoed in a recent in vivo dissection of Drosophila epsin that also revealed the importance of the UIM in Notch signaling (Xie et al., 2012), as well as in previous studies that implicate a critical role for the UIM in receptor transport (Shih et al., 2002; Overstreet et al., 2003; Sugiyama et al., 2005; Dores et al., 2009; Kazazic et al., 2009). However, the mechanistic role for the UIM remains unclear. "
    [Show abstract] [Hide abstract]
    ABSTRACT: Low density lipoprotein receptor (LDLR) internalization clears cholesterol-laden LDL particles from circulation in humans. Defects in clathrin-dependent LDLR endocytosis promote elevated serum cholesterol levels and can lead to atherosclerosis. However, our understanding of the mechanisms that control LDLR uptake remains incomplete. To identify factors critical to LDLR uptake, we pursued a genome-wide RNAi screen using Caenorhabditis elegans LRP-1/megalin as a model for LDLR transport. In doing so, we discovered an unanticipated requirement for the clathrin-binding endocytic adaptor epsin1 in LDLR endocytosis. Epsin1 depletion reduced LDLR internalization rates in mammalian cells, similar to that observed following clathrin depletion. Genetic and biochemical analyses of epsin in C. elegans and mammalian cells uncovered a requirement for the ubiquitin-interaction motif (UIM) as critical for receptor transport. As the epsin UIM promotes the internalization of some ubiquitinated receptors, we predicted LDLR ubiquitination as necessary for endocytosis. However, engineered ubiquitination-impaired LDLR mutants showed modest internalization defects that were further enhanced with epsin1 depletion, demonstrating epsin1-mediated LDLR endocytosis is independent of receptor ubiquitination. Finally, we provide evidence that epsin1-mediated LDLR uptake occurs independently of either of the two documented internalization motifs (FxNPxY or HIC) encoded within the LDLR cytoplasmic tail, indicating an additional internalization mechanism for LDLR.
    Molecular biology of the cell 12/2012; 24(3). DOI:10.1091/mbc.E12-02-0163 · 4.47 Impact Factor
  • Source
    • "We found previously [32] that either full-length LqfRa fused at its C-terminus to GFP (LqfRaFL-GFP) or a version of the fusion protein that lacks the ENTH domain (LqfRaΔENTH -GFP), when expressed using Gal4/UAS and the ubiquitous Actin5C-gal4 driver, is sufficient to rescue all of the obvious defects due to loss of lqfR+ gene activity: these include larval lethality and the absence of imaginal discs. The dispensability of the ENTH domain was not entirely surprising, as endocytic Epsin also functions well without its ENTH domain [33], [34]. However, further structure/function experiments did yield results that were completely unexpected. "
    [Show abstract] [Hide abstract]
    ABSTRACT: Tel2, a protein conserved from yeast to vertebrates, is an essential regulator of diverse cellular processes including telomere maintenance, DNA damage checkpoints, DNA repair, biological clocks, and cell signaling. The Drosophila Tel2 protein is produced as a translational fusion with EpsinR, a Clathrin adapter that facilitates vesicle trafficking between the Golgi and endosomes. EpsinR and Tel2 are encoded by a Drosophila gene called lqfR. lqfR is required for viability, and its specific roles include cell growth, proliferation, and planar cell polarity. We find that all of these functions of lqfR are attributed entirely to Tel2, not EpsinR. In addition, we find that Drosophila LqfR/Tel2 is a component of one or more protein complexes that contain E-cadherin and Armadillo. Moreover, Tel2 modulates E-cadherin and Armadillo cellular dynamics. We propose that at least one of the functions of Drosophila Tel2 is regulation of Wingless signaling.
    PLoS ONE 09/2012; 7(9):e46357. DOI:10.1371/journal.pone.0046357 · 3.23 Impact Factor
  • Source
    [Show abstract] [Hide abstract]
    ABSTRACT: Notch signaling induced by cell surface ligands is critical to development and maintenance of many eukaryotic organisms. Notch and its ligands are integral membrane proteins that facilitate direct cell-cell interactions to activate Notch proteolysis and release the intracellular domain that directs Notch-specific cellular responses. Genetic studies suggest that Notch ligands require endocytosis, ubiquitylation, and epsin endocytic adaptors to activate signaling, but the exact role of ligand endocytosis remains unresolved. Here we characterize a molecularly distinct mode of clathrin-mediated endocytosis requiring ligand ubiquitylation, epsins, and actin for ligand cells to activate signaling in Notch cells. Using a cell-bead optical tweezers system, we obtained evidence for cell-mediated mechanical force dependent on this distinct mode of ligand endocytosis. We propose that the mechanical pulling force produced by endocytosis of Notch-bound ligand drives conformational changes in Notch that permit activating proteolysis.
    Developmental Cell 05/2012; 22(6):1299-312. DOI:10.1016/j.devcel.2012.04.005 · 9.71 Impact Factor
Show more